The NIDDK High School Short-Term Research Experience for Underrepresented Persons.

Background: Increasing the pipeline of aspiring minority biomedical/health professionals is a crucial component to diversifying the health science workforce. The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) created the High School Short-Term Research Experience for Underrepresented Persons (HS-STEP-UP) to provide introductory biomedical/biobehavioral research experiences to promising high school students, who are traditionally underrepresented in the biomedical/biobehavioral sciences. The program reaches out to African American and Lationo/Hispanic students, as well as Native American students and students from the United States Territories. Methods: HS-STEP-UP provides a stimulating, rigorous 8- to 10-week summer research experience for a national cohort of ~100 high school students each year; the experience is organized through four National Institutes of Health (NIH)-funded coordinating centers. Typically, the program receives about 300 applications a year and about 100 students are accepted. Applicants are reviewed and selected based upon their online application that includes: a high school transcript, list of classes and extracurricular activities, two recommendation letters and a personal statement. The program culminates with a symposium at the NIH where students present their research and attend workshops and seminars. Results: For the 2017 and 2018 HS-STEP-UP programs, the classes included 193 students; 67% were females and 82% were underrepresented minorities. Forty eight percent of students reported a family income <$37,000/year, and 23% were from first generation college families. Ninety percent were very satisfied or satisfied with their research topic and 94% rated the end of the year symposium at NIH as excellent or very good. Only 65% were very satisfied or satisfied with their mentor matching, and 21% stated they were dissatisfied or very dissatisfied with their mentor. All the students successfully completed their summer research projects and presented their research abstracts at the symposium. All participating seniors reported attending college. Conclusion: HS-STEP-UP has been highly successful in recruiting traditionally underrepresented students and supporting underrepresented HS students with a rewarding introductory experience to research. Students are overall satisfied with the program, but mentor matching needs more attention. Longer-term follow-up is needed to determine how participating in STEP UP impacts their decisions to participate in the biomedical workforce in the future.

GNAI1 and GNAI3 Reduce Colitis-Associated Tumorigenesis in Mice by Blocking IL6 Signaling and Down-regulating Expression of GNAI2.

BACKGROUND & AIMS: Interleukin 6 (IL6) and tumor necrosis factor contribute to the development of colitis-associated cancer (CAC). We investigated these signaling pathways and the involvement of G protein subunit alpha i1 (GNAI1), GNAI2, and GNAI3 in the development of CAC in mice and humans. METHODS: B6;129 wild-type (control) or mice with disruption of Gnai1, Gnai2, and/or Gnai3 or conditional disruption of Gnai2 in CD11c+ or epithelial cells were given dextran sulfate sodium (DSS) to induce colitis followed by azoxymethane (AOM) to induce carcinogenesis; some mice were given an antibody against IL6. Feces were collected from mice, and the compositions of microbiomes were analyzed by polymerase chain reactions. Dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) isolated from spleen and colon tissues were analyzed by flow cytometry. We performed immunoprecipitation and immunoblot analyses of colon tumor tissues, MDSCs, and mouse embryonic fibroblasts to study the expression levels of GNAI1, GNAI2, and GNAI3 and the interactions of GNAI1 and GNAI3 with proteins in the IL6 signaling pathway. We analyzed the expression of Gnai2 messenger RNA by CD11c+ cells in the colonic lamina propria by PrimeFlow, expression of IL6 in DCs by flow cytometry, and secretion of cytokines in sera and colon tissues by enzyme-linked immunosorbent assay. We obtained colon tumor and matched nontumor tissues from 83 patients with colorectal cancer having surgery in China and 35 patients with CAC in the United States. Mouse and human colon tissues were analyzed by histology, immunoblot, immunohistochemistry, and/or RNA-sequencing analyses. RESULTS: GNAI1 and GNAI3 (GNAI1;3) double-knockout (DKO) mice developed more severe colitis after administration of DSS and significantly more colonic tumors than control mice after administration of AOM plus DSS. Development of increased tumors in DKO mice was not associated with changes in fecal microbiomes but was associated with activation of nuclear factor (NF) κB and signal transducer and activator of transcription (STAT) 3; increased levels of GNAI2, nitric oxide synthase 2, and IL6; increased numbers of CD4+ DCs and MDSCs; and decreased numbers of CD8+ DCs. IL6 was mainly produced by CD4+/CD11b+, but not CD8+, DCs in DKO mice. Injection of DKO mice with a blocking antibody against IL6 reduced the expansion of MDSCs and the number of tumors that developed after CAC induction. Incubation of MDSCs or mouse embryonic fibroblasts with IL6 induced activation of either NF-κB by a JAK2-TRAF6-TAK1-CHUK/IKKB signaling pathway or STAT3 by JAK2. This activation resulted in expression of GNAI2, IL6 signal transducer (IL6ST, also called GP130) and nitric oxide synthase 2, and expansion of MDSCs; the expression levels of these proteins and expansion of MDSCs were further increased by the absence of GNAI1;3 in cells and mice. Conditional disruption of Gnai2 in CD11c+ cells of DKO mice prevented activation of NF-κB and STAT3 and changes in numbers of DCs and MDSCs. Colon tumor tissues from patients with CAC had reduced levels of GNAI1 and GNAI3 and increased levels of GNAI2 compared with normal tissues. Further analysis of a public human colorectal tumor DNA microarray database (GSE39582) showed that low Gani1 and Gnai3 messenger RNA expression and high Gnai2 messenger RNA expression were significantly associated with decreased relapse-free survival. CONCLUSIONS: GNAI1;3 suppresses DSS-plus-AOM-induced colon tumor development in mice, whereas expression of GNAI2 in CD11c+ cells and IL6 in CD4+/CD11b+ DCs appears to promote these effects. Strategies to induce GNAI1;3, or block GNAI2 and IL6, might be developed for the prevention or therapy of CAC in patients.

Antibody and T cell responses in reciprocal prime-boost studies with full-length and truncated merozoite surface protein 1-42 vaccines.

The P. falciparum Merozoite Surface Protein 1-42 (MSP1-42) is one of the most studied malaria subunit vaccine candidates. The N-terminal fragment of MSP1-42, MSP1-33, is primarily composed of allelic sequences, and has been shown to possess T helper epitopes that influence protective antibody responses toward the C-terminal region, MSP1-19. A truncated MSP1-42 vaccine, Construct 33-I, consisting of exclusively conserved T epitope regions of MSP1-33 expressed in tandem with MSP1-19, was previously shown to be a more effective immunogen than the full-length MSP1-42 vaccine. Here, by way of reciprocal priming/boosting immunization regimens, we studied the immunogenicity of Construct 33-I in the context of recognition by immune responses induced by the full-length native MSP1-42 protein, in order to gauge the effects of pre- and post-exposures to MSP1-42 on vaccine induced responses. Judging by immune responsiveness, antibody and T cell responses, Construct 33-I was effective as the priming antigen followed by full-length MSP1-42 boosting, as well as the boosting antigen following full-length MSP1-42 priming. In particular, Construct 33-I priming elicited the broadest responsiveness in immunized animals subsequently exposed to MSP1-42. Moreover, Construct 33-I, with its conserved MSP1-33 specific T cell epitopes, was equally well recognized by homologous and heterologous allelic forms of MSP1-42. Serum antibodies raised against Construct 33-I efficiently inhibited the growth of parasites carrying the heterologous MSP1-42 allele. These results suggest that Construct 33-I maintains and/or enhances its immunogenicity in an allelic or strain transcending fashion when deployed in populations having prior or subsequent exposures to native MSP1-42s.

Iron oxide nanoparticles as a clinically acceptable delivery platform for a recombinant blood-stage human malaria vaccine.

This study explored the novel use of iron oxide (IO) nanoparticles (<20 nm) as a vaccine delivery platform without additional adjuvants. A recombinant malaria vaccine antigen, the merozoite surface protein 1 (rMSP1), was conjugated to IO nanoparticles (rMSP1-IO). Immunizations in outbred mice with rMSP1-IO achieved 100% responsiveness with antibody titers comparable to those obtained with rMSP1 formulated with a clinically acceptable adjuvant, Montanide ISA51 (2.7×10 vs. 1.6×10; respectively). Only rMSP1-1O could induce significant levels (80%) of parasite inhibitory antibodies. The rMSP1-IO was highly stable at 4°C and was amenable to lyophilization, maintaining its antigenicity, immunogenicity, and ability to induce inhibitory antibodies. Further testing in nonhuman primates, Aotus monkeys, also elicited 100% immune responsiveness and high levels of parasite inhibitory antibodies (55-100% inhibition). No apparent local or systemic toxicity was associated with IO immunizations. Murine macrophages and dendritic cells efficiently (>90%) internalized IO nanoparticles, but only the latter were significantly activated, with elevated expression/secretion of CD86, cytokines (IL-6, TNF-α, IL1-b, IFN-γ, and IL-12), and chemokines (CXCL1, CXCL2, CCL2, CCL3, CCL4, and CXCL10). Thus, the IO nanoparticles is a novel, safe, and effective vaccine platform, with built-in adjuvancy, that is highly stable and field deployable for cost-effective vaccine delivery.

Blood stage merozoite surface protein conjugated to nanoparticles induce potent parasite inhibitory antibodies.

In this proof-of-concept study we report the use of <15 nm, water soluble, inorganic nanoparticles as a vaccine delivery system for a blood stage malaria vaccine. The recombinant malarial antigen, Merozoite Surface Protein 1 (rMSP1) of Plasmodium falciparum served as the model vaccine. The rMSP1 was covalently conjugated to polymer-coated quantum dot CdSe/ZnS nanoparticles (QDs) via surface carboxyl groups, forming rMSP1-QDs. Anti-MSP1 antibody responses induced by rMSP1-QDs were found to have 2-3 log higher titers than those obtained with rMSP1 administered with the conventional adjuvants, Montanide ISA51 and CFA. Moreover, the immune responsiveness and the induction of parasite inhibitory antibodies were significantly superior in mice injected with rMSP1-QDs. The rMSP1-QDs delivered via intra-peritoneal (i.p.), intra-muscular (i.m.), and subcutaneous (s.c.) routes were equally efficacious. The high level of immunogenicity exhibited by the rMSP1-QDs was achieved without further addition of other adjuvant components. Bone marrow derived dendritic cells were shown to efficiently take up the nanoparticles leading to their activation and the expression/secretion of key cytokines, suggesting that this may be a mode of action for the enhanced immunogenicity. This study provides promising results for the use of water soluble, inorganic nanoparticles (<15 nm) as potent vehicles/platforms to enhance the immunogenicity of polypeptide antigens in adjuvant-free immunizations.

T cell epitope regions of the P. falciparum MSP1-33 critically influence immune responses and in vitro efficacy of MSP1-42 vaccines.

The C-terminal 42 kDa fragments of the P. falciparum Merozoite Surface Protein 1, MSP1-42 is a leading malaria vaccine candidate. MSP1-33, the N-terminal processed fragment of MSP1-42, is rich in T cell epitopes and it is hypothesized that they enhance antibody response toward MSP1-19. Here, we gave in vivo evidence that T cell epitope regions of MSP1-33 provide functional help in inducing anti-MSP1-19 antibodies. Eleven truncated MSP1-33 segments were expressed in tandem with MSP1-19, and immunogenicity was evaluated in Swiss Webster mice and New Zealand White rabbits. Analyses of anti-MSP1-19 antibody responses revealed striking differences in these segments' helper function despite that they all possess T cell epitopes. Only a few fragments induced a generalized response (100%) in outbred mice. These were comparable to or surpassed the responses observed with the full length MSP1-42. In rabbits, only a subset of truncated antigens induced potent parasite growth inhibitory antibodies. Notably, two constructs were more efficacious than MSP1-42, with one containing only conserved T cell epitopes. Moreover, another T cell epitope region induced high titers of non-inhibitory antibodies and they interfered with the inhibitory activities of anti-MSP1-42 antibodies. In mice, this region also induced a skewed TH2 cellular response. This is the first demonstration that T cell epitope regions of MSP1-33 positively or negatively influenced antibody responses. Differential recognition of these regions by humans may play critical roles in vaccine induced and/or natural immunity to MSP1-42. This study provides the rational basis to re-engineer more efficacious MSP1-42 vaccines by selective inclusion and exclusion of MSP1-33 specific T cell epitopes.

ICU nurses' perceptions of potential constraints and anticipated support to practice defibrillation: a qualitative study.

AIM: The study examines the experience of intensive care nurses in caring for patients in cardiac arrest, and their perceptions of introducing nurse-led defibrillation. METHOD: This was a descriptive, exploratory and qualitative study at an intensive care unit (ICU) of an acute regional hospital in Hong Kong. Twelve registered nurses were purposefully selected for interview. RESULTS: Although all the participants were trained in basic life support, only 50% were trained in advanced cardiac life support (ACLS), and those trained in ACLS described having limited opportunities to apply their defibrillation knowledge. Whilst participants believed that they were theoretically prepared to influence the patient's resuscitation outcomes, newly qualified nurses were reluctant to be accountable for defibrillation. In contrast, experienced nurses were more willing to perform nurse-led defibrillation. Support from management, cooperation between nurses and doctors, regular in-hospital 'real-drill' programmes, sponsorship for training, and the use of alternative defibrillation equipment should be considered to encourage nurse-led defibrillation in ICU settings. CONCLUSION: Nurse-led defibrillation is an approach of delivering prompt care to critically ill patients, and a way ahead for intensive care nursing in Hong Kong. Emphasis on a consistent policy to promote nurse-led defibrillation practice is needed.

Biological activities of anti-merozoite surface protein-1 antibodies induced by adjuvant-assisted immunizations in mice with different immune gene knockouts.

Immunizations with Plasmodium falciparum MSP1-42 or MSP1-19 induce antibodies that inhibit parasites in vitro, which correlates with in vivo protective immunity by vaccination. We previously showed that several adjuvant formulations can induce anti-MSP1-19 antibodies in interleukin-6, intercellular adhesion molecule 1, CD80, and CD86 knockout (KO) mice and at levels similar to those obtained in the healthy uninfected hosts. Here, we determine whether these immune gene KOs or the immunopotentiating activities of the adjuvants have a more important influence on the induction of parasite-inhibitory anti-MSP1-19 antibodies. Results showed that the biological activities of the anti-MSP1-19 antibodies induced by these adjuvants were not affected by the immune gene KOs. All adjuvant formulations that induced significant inhibitory antibody responses (i.e., >50% inhibition of parasite growth) contained monophosphoryl lipid A (MPL) in emulsion carriers, whereas MPL or emulsion carriers alone were ineffective. The ability to retain vaccine efficacy by the MSP1-19 and adjuvant formulations in the altered immunological background is a valuable and significant attribute in light of many instances of skewed immune status in the targeted vaccine populations.

Adjuvant formulations possess differing efficacy in the potentiation of antibody and cell mediated responses to a human malaria vaccine under selective immune genes knockout environment.

Infections and chronic diseases can alter the host's immunological balance or result in immunodeficiencies. We hypothesize that this may also affect the performance of vaccine adjuvants. Accordingly, the potency and adjuvanticity of eight adjuvant formulations based on Montanide ISA720, MF59, monophosphoryl lipid A (MPL), QS21 (saponin derivative), MPL-SE (stable emulsion of a MPL derivative), and MPL-AF (MPL in aqueous formulation) were studied in immune gene knockout mice, IFN-gamma -/-, IL-4 -/-, and STAT6 -/-, using the P. falciparum MSP1 vaccine, P30P2MSP1-19 as a model immunogen. The adjuvants showed preferential requirements for the immune mediators to induce immune responses to MSP1-19, and the effects were formulation-specific. While emulsion-type adjuvants were highly effective in mice, their potency was more readily suppressed by immune knockouts; and additions of immunomodulators were required to restore efficacy. Formulated adjuvants had characteristics distinct from their individual components, and multi-components formulations were not necessarily superior. We conclude that perturbation of immune environments will have measurable impact on adjuvants' potency. Evaluation of adjuvants in immune knockout models may be a supplementary approach to measure and compare adjuvants' efficacy, and to further unveil their distinct biological activities.

The requirement of CD80, CD86, and ICAM-1 on the ability of adjuvant formulations to potentiate antibody responses to a Plasmodium falciparum blood-stage vaccine.

Many adjuvants are known to enhance expression of co-stimulatory and adhesion molecules secondarily to the activation of immune cells. Whether interactions via these molecules are obligatory in adjuvants' ability to potentiation vaccine immunogenicity is less clear. We investigated the ability of eight adjuvant formulations to potentiate the immunogenicity of a malaria vaccine in mice deficient in the prominent co-stimulatory molecules, CD80 and CD86; and the adhesion ligand, ICAM-1. While no adjuvants could bypass co-stimulatory requirements, more formulations exhibited dependency for CD86 than for CD80. In CD80 or CD86 KO mice, formulations with the saponin derivative, QS21 could efficiently default to the other B7 molecule. This effect was dominant over other adjuvant constituents. The requirement for ICAM-1 could be readily bypassed using adjuvant formulations containing immunomodulators; whereas this was not the case with emulsion-type adjuvants in which reduction in adjuvanticity was associated with decreases in antigen-specific IFN-gamma responses. These studies may help to guide the formulation of vaccine adjuvants to maintain effectiveness in hosts with altered immunological environment that often result from infections.

Interleukin-6 has differential influence on the ability of adjuvant formulations to potentiate antibody responses to a Plasmodium falciparum blood-stage vaccine.

The efficacy of vaccine adjuvants can be influenced by the immunological environment of the host, depending on the mechanism(s) by which they exert their immunopotentiating activities. Interleukin-6 is a pleiotropic cytokine that has a broad range of biological activities on immune and non-immune cells. We investigated the role of IL-6 on the ability of nine adjuvant formulations to induce antibody responses to the Plasmodium falciparum MSP1-19 malaria vaccine, using IL-6-/- (KO) mice. Results showed that some adjuvants, i.e. MPL-SE, CFA/IFA, ISA720/QS21/MPL, depended on IL-6 for their efficacy, while others exhibited increased potency in its absence. The efficacy of adjuvants in the IL-6 KO environment cannot be solely attributed to their ability to stimulate antigen-specific cellular responses, suggesting that other biological activities of IL-6 are also important. The results further suggest that two adjuvants utilized dissimilar pathways to potentiate the same type of immune response.

Antigenicity and immunogenicity of the N-terminal 33-kDa processing fragment of the Plasmodium falciparum merozoite surface protein 1, MSP1: implications for vaccine development.

The Plasmodium falciparum merozoite surface protein 1 (MSP1), MSP1-42 and MSP1-19 are protective malaria vaccines. MSP1-42 is cleaved to form MSP1-33 and MSP1-19. The role of MSP1-33 in immunity is unclear. We investigated the antibody responses to MSP1-33; and to MSP1-33Trunc, in which major conserved sequences were excised. While anti-MSP1-33 antibodies were subdominant in the anti-MSP1-42 responses, immunizations with MSP1-33 or MSP1-33Trunc induced high levels of antibodies reactive with MSP1-42 or whole merozoites. Anti-MSP1-33 and anti-MSP1-33Tunc antibodies crossreacted with both allelic forms of MSP1-42. Anti-MSP1-33 sera were ineffective in inhibiting parasite growth in vitro; but they significantly enhanced the activities of sub-optimal concentrations of the inhibitory anti-MSP1-42 sera. Thus, immunization strategies with MSP1-based vaccines may benefit from co-induction of anti-MSP1-33 responses to enhance efficacy and potency.

Plasmodium falciparum anti-MSP1-19 antibodies induced by MSP1-42 and MSP1-19 based vaccines differed in specificity and parasite growth inhibition in terms of recognition of conserved versus variant epitopes.

The C-terminal 42 kDa fragment (MSP1-42) and its smaller 19 kDa subfragment (MSP1-19) of the Plasmodium falciparum merozoite surface protein, MSP1, are leading candidate malaria vaccines. Since the targets of protective immunity lie within the MSP1-19, we compared the anti-MSP1-19 antibodies induced by vaccination with recombinant MSP1-42 and MSP1-19. The specificities of the antibody responses were analyzed using five recombinant MSP1-19s expressing different naturally occurring variant amino acid residues. We observed dramatic differences in the specificities of the anti-MSP1-19 antibodies induced by the two vaccines. MSP1-42 consistently induced crossreactive antibodies; whereas the antibodies induced by recombinant MSP1-19 were highly variable among animals in terms of recognition of conserved versus variant epitopes. Of the variant residues examined, only a subset significantly contributed as part of immunogenic B epitopes. MSP1-42 consistently induced potent growth inhibitory antibodies that recognized conserved epitopes, leading to efficient inhibition of heterologous parasites. In contrast, MSP1-19 induced strong inhibitory antibody responses in only a subset of animals studied. In some of the MSP1-19 immunized animals, inhibition of homologous parasites may be due to recognition of inhibitory epitopes associated with the homologous variant residues, and the induction of antibodies to conserved inhibitory epitopes may not be efficiently achieved. These data suggest an advantage of using MSP1-42 over MSP1-19 based vaccines.

Plasmodium falciparum: immunization with MSP1-42 induced non-inhibitory antibodies that have no blocking activities but enhanced the potency of inhibitory anti-MSP1-42 antibodies.

Hyperimmunization with Plasmodium falciparum MSP1-42 could induce antibodies that have little or no parasite growth inhibitory activities. These antisera had no blocking activities as determined by their ability to interfere with the in vitro activities of growth inhibitory anti-MSP1-42 sera. Equally important, they enhanced the potency of growth inhibitory anti-MSP1-42 sera.

In vivo expression and immunological studies of the 42-kilodalton carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein 1 in the baculovirus-silkworm system.

The 42-kDa carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein 1 (MSP-1(42)) is an anti-erythrocytic stage malaria vaccine candidate. In this study, MSP-1(42) was expressed by using the Bombyx mori nuclear polyhedrosis virus-silkworm expression system, and the antigenicity and immmunogenicity of the recombinant protein, Bmp42, were evaluated. The average yield of Bmp42, as determined by a sandwich enzyme-linked immunosorbent assay (ELISA), was 379 microg/ml of infected silkworm hemolymph, which was >100-fold higher than the level attainable in cell culture medium. N-terminal amino acid sequencing revealed that Bmp42 was correctly processed in silkworm cells. Data from immunoblotting, as well as from the inhibition ELISA, suggested that the conformational B-cell epitopes of MSP-1(42) were recreated in Bmp42. Immunization of rabbits with Bmp42 in complete Freund's adjuvant generated high-titer antibody responses against the immunogen. Specificity analyses of the anti-Bmp42 antibodies using several recombinant MSP-1(19) proteins expressing variant and conserved B-cell epitopes suggested that the anti-Bmp42 antibodies recognized primarily conserved epitopes on MSP-1(19). Furthermore, the anti-Bmp42 antibodies were highly effective in inhibiting the in vitro growth of parasites carrying homologous or heterologous MSP-1(42). Our results demonstrated that the baculovirus-silkworm expression system could be employed to express biologically and immunologically active recombinant MSP-1(42) at elevated levels; thus, it is an attractive alternative for producing a protective MSP-1(42) vaccine for human use.